Contrast Agents for Magnetic Resonance Imaging
Accurate detection of abnormalities in a patient's body is an essential prerequisite for diagnosing and adequately treating disease. Visualization methods, e.g., magnetic resonance imaging (MRI), are becoming more important for such accurate detection. MRI is non-invasive and requires no exposure of humans to potentially harmful radiation. In MRI, tissues of different origin, such as normal and deviated, e.g., cancerous, tissues, may be differentiated on the basis of differences in relaxation times T1, the spin-lattice or longitudinal relaxation time, or T2, the spin-spin or transverse relaxation time. Because of these differences, differential signal intensity is produced which gives various degrees of contrast in MR images. The greater the difference in T1 or T2, the more pronounced the contrast. However, in many cases diseased or deviated tissue is isointensive, i.e., the diseased or deviated tissue has the same signal intensity as normal tissue, and is therefore not distinguishable from normal tissue without the use of special contrast agents.
Where MR imaging techniques employed to elucidate blood perfusion defects are based on the differentiation of flowing blood from stationary surrounding tissues, e.g., MR angiography (MRA). Three dimensional angiographic techniques, e.g., "Time of Flight" (TOF) and "Phase Contrast" (PC) techniques, provide detailed images of intracranial vessels. However, traditional MRA, i.e., Time of Flight MRA, is dependent on flow velocity and flow shape and thus high-quality angiography of peripheral vessels with high flow resistance is generally impossible due to an effect known as vessel saturation. To overcome this problem, contrast agents have been used to selectively lower the relaxation times of blood.
Gadolinium (III) diethylenetriamine pentaacetic acid (Gd-DTPA) dimeglumine is a widely used contrast agent which is relatively small (MW538) and extravasates on the first pass through the capillaries. However, the use of Gd-DTPA for MR angiography in all organs except the brain is limited, since the blood half-life of Gd-DTPA is less than 20 minutes, and the biological life in man of GD-DTPA is about 90 minutes. The extravasation results in a rapid decrease in vessel/muscle signal ratio, which makes the accurate detection of abnormalities and disease difficult. Moreover, Gd-DTPA dimeglumine, which is used in clinical practice, is immunogenic, which does not favor its repetitious administration to the same patient.
Similar problems occur with the use of ferrioxamine-B as a contrast agent. In addition, ferrioxamine-B causes a precipitous drop in blood pressure after its intravenous administration.
MRI contrast agents created using natural and synthetic macromolecules offer the advantage of high molecular relaxivity due to the multiple chelating groups coupled to a single polymer backbone. These groups can chelate paramagnetic cations, e.g., in Gd-DTPA-poly-l-lysine, or produce high relaxivity due to the presence of iron oxide, e.g., in iron-containing colloids. However, iron oxide-based colloids have their own ligand-independent specific site of accumulation in the body, e.g., the liver, spleen, and lymphoid tissues.
Chelating groups may be attached to a variety of natural polymers, e.g., proteins and polysaccharides, and synthetic polymers. Chemical attachment, e.g., by conjugation, of DTPA to bovine serum albumin will result in a macromolecular contrast agent, which is suitable for some applications, e.g., NMR-angiography, but because of the efficient recognition of modified albumin by macrophages, and albumin-receptors on endothelial cells this contrast agent has a short blood half-life. It is also immunogenic and toxic to reticuloendothelial system organs. Therefore, use for MR imaging is limited.
One way to diminish the antigenicity of albumin is to mask it with natural and synthetic polymers, e.g., spacer arms, by covalent attachment, but this leaves few reactive groups in the protein globule which are needed for binding the chelates and paramagnetic cations. Therefore, the use of such complexes in MR imaging is limited.
Synthetic polymers of 1-amino acids, such as poly-l-lysine (PL), are an alternative to modified natural proteins as backbones for contrast agents. PL modified with DTPA can be used as a radionuclide carrier for antibody-mediated targeting in nuclear medicine. Poly-1-lysine-DTPA, i.e., poly-l-lysine with DTPA groups bonded to epsilon-amino groups of lysine residues has been suggested as a Gd complexone, i.e., a compound which forms a complex with Gd, for use in MR angiography. It is also known that the toxicity of DTPA-poly-l-lysine is lower than that of DTPA-albumin. However, DTPA-moieties on DTPA-polylysine are recognized by liver Kupffer cells and some kidneys cells, presumably glomerulonephral phagocytes, which cause elevated and relatively rapid removal of the contrast agent from the blood. For example, 90% of the intravenously injected agent, e.g., poly-l-lysine-DTPA(Gd) (MW 48.7 kD), is removed from circulation in 1 hour (t.sub. 1/2 =0.134 h) and accumulated in the kidneys, liver, and bone. Moreover, synthesis of DTPA-poly-l-lysine can be carried out with a cross-linking reagent, e.g., cyclic anhydride of DTPA. As a result, it is difficult to avoid the formation of cross-linked products of relatively high molecular weight and the preparation obtained is heterogeneous.
Nitrogen-containing polymers, e.g., polethyleneimine, have been modified with monofunctional derivatives of acetic acid to form a molecule where the backbone nitrogens and acetic acid residues are involved in complex formation with trivalent cations. However, because of extensive undesirable accumulation in the liver, paramagnetic complexes of polyethyleneiminoacetic acid are not widely used in MRI.
Polymeric contrast agents, e.g., starburst dendrimers, constitute a separate family of macromolecules with limited potential value as contrast agents. This family of agents has not been shown to be biocompatible and thus its value for in vivo imaging is limited.
Various polysaccharide-based chelating agents have been previously described; however, their activation complement which has been shown to be a feature of polysaccharides, preclude their extensive use in MR imaging.
Agents with Extended Blood Half-Life
Blood half-life and immunogenicity are crucial characteristics of any contrast agent designed for therapy or medical diagnosis. In some cases, such as enzyme-replacement therapy, fast elimination of therapeutic agents from circulation and accumulation in antigen-presenting cells limit their potential use in the treatment of disease. To overcome this problem, it has been suggested to chemically modify the macromolecular agents, e.g., enzymes, with various natural and synthetic polymers. Dextrans, synthetic polyamino acids, and polyethylene glycols are used most frequently. However, only polyethylene glycol (PEG) and its monomethyl ester (MPEG) are suitable to prolong blood half-life and simultaneously decrease the immunogenicity of the therapeutic agent. The reason for modifying antigenic determinants by MPEG may be explained by the screening of electrostatic charge of the protected micromolecule, e.g., protein, and by the ability to form numerous bonds with water in solutions.
About three molecules of water are associated with each ethylene oxide unit and form the immediately adjacent water microenvironment for the polymer. This prevents, to a great extent, the adsorptive interactions of proteins and cells with PEG chains. The use of PEG in its activated forms, e.g., 4,6-dichloro-s-triazine-activated PEG or MPEG, is undesirable for protein modification, because the activated product is contaminated with by-products and is highly moisture-sensitive. Stable and virtually non-biodegradable biodegradable bonds have been formed by the conjugation of MPEG, e.g., reacting 4,6-dichloro-s-triazine and 1,1'-carbonyldiimidazole with aminogroups.
PEG and MPEG are used in contrast agents for medical imaging. Covalent modifications of desferrioxamine-B with MPEG improve the body's tolerance of such contrast agents in vivo, but does not result in any significant change in imaging efficacy. Contrast agents containing MPEG or PEG as a component of paramagnetic mixtures or in cross-linked paramagnetic polymers also have been used.
Targeted Contrast Agents
Contrast agents targeted to the sites of interest help to increase the effectiveness of MR imaging methods. Such diagnostic agents may include combinations of a ligand and a paramagnetic contrast agent coupled by strong interaction, e.g., a covalent chemical bond. After systemic application, such a contrast agent accumulates in the target site which is determined by ligand specificity. As a result, the site of accumulation is easily differentiated from surrounding tissue because it appears hyper- (or hypo-) intensive on MR images. The ligand which directs the contrast agent to the target site may be specific to receptors on either normal or transformed cells of a given organ or tissue. In the first case the contrast agent will be accumulated in normal tissue; in the second case, it will be accumulated in altered tissue.
Success in designing a targeted contrast agent is mainly determined by the following properties: 1. avidity to target site; 2. antigenicity, i.e., ability to pass through capillary endothelium; and 3. blood half-life of the ligand or targeting ("vector") molecule. Coupling a contrast agent to a targeting ligand molecule, e.g., an antibody or its fragments, which creates a targeted contrast agent, e.g., a chelated paramagnetic cation, paramagnetic colloid or combination of a chelate and a paramagnetic colloid conjugated to a targeting molecule, typically decreases its potential value for any of a number of reasons, e.g., decreased avidity to a target site, increased antigenicity, or decreased half-life. For example, coupling of a small antibody fragment, e.g., a Fab or Fv chimeric molecule, to a large paramagnetic molecule, e.g., DTPA-polymer, or a superparamagnetic colloid, e.g., iron oxide, to form a targeted contrast agent will increase the immune response of the recipient organism to the agent because of the adjuvant properties of the agent itself. The paramagnetic molecule or colloid itself may be recognized by the recipient organism's opsonizing proteins and the contrast agent may be trapped in reticuloendothelial system organs. As a result, the contrast agent is removed from the circulation by the liver and spleen before any substantial concentration is achieved in the target site. Moreover, such a contrast agent may be recognized as a foreign antigen which may give rise to undesirable host antibodies.